Wnt proteins are phylogenetically conserved, secreted glycoproteins that regulate cell-to-cell communication during embryonic development and adult tissue homeostasis through canonical and non-canonical signaling pathways. Wnt signaling is intensively studied due to its central role, and therapeutic implications, in the development and progression of cancer, tissue renewal, and differentiation of stem cells. Wnt binding to Frizzled, which has structural similarities to G-protein coupled receptors, and the co-receptors Lrp5/6, is indispensable for Wnt/-catenin signaling, and is counterbalanced by a variety of endogenous antagonists. In this proposal we address the complete lack of extracellular structural information on Wnts, the manner in which Wnts complex with Frizzled receptors, as well as Wnt and Frizzled interactions with endogenous inhibitory proteins such as WIF, Dkk and Kremen. Given the central importance of Wnt signaling for a variety of human diseases, structural information has now become critical in order to clearly delineate the basis of receptor- ligand specificity and mechanisms of receptor activation. A current confusion in this field is that Wnts, Fz and Lrp6 are highly cross-reactive, complicating the attribution of specific biological phenotypes to specific Wnt-Fz receptor pairs. Structures of Wnt-receptor complexes could help solve this problem by visualizing degenerate versus ligand-specific contacts, as well as the architectures of the higher order signaling complexes. However, a major technical obstacle to obtaining structural data has been that Wnt proteins contain a hydrophobic lipid modification that complicates Wnt over-expression and crystallization. In this proposal we utilize a variety of novel (yeast display) and traditional (X-ray crystallography) methodologies to obtain three-dimensional structures of extracellular Wnt complexes involved in receptor activation, and inhibition. We aim to reconstitute several recombinant Wnt-Frizzled, Wnt-Frizzled-Lrp5/6 and Wnt-antagonist complexes, in order to characterize their assemblies, affinities and stoichiometries. We will also attempt a highly innovative experiment to produce water-soluble, non-palmitoylated, bioactive Wnts by in vitro evolution. Using X-ray crystallography, we hope to elucidate the structural principles of Wnt-Frizzled-Lrp5/6 recognition, and inhibition of Wnt-Frizzled-Lrp5/6 complex formation by the antagonists WIF, DKK and Kremen, and together with established collaborators in the Wnt field, translate the structural data into functionally insightful experiments. Ultimately the results of these ambitious studies will be valuable for revealing new protein engineering strategies to interrogate and therapeutically target Wnt signaling.